1Biofilms are community structures of bacteria enmeshed in a self-produced matrix of exopolysaccharides.
2The biofilm matrix serves numerous roles, including resilience and persistence, making biofilms a subject 3 of research interest among persistent clinical pathogens of global health importance. Our current 4 understanding of the underlying biochemical pathways responsible for biosynthesis of these 5 exopolysaccharides is largely limited to Gram-negative bacteria. Clostridia are a class of Gram-positive, 6 anaerobic and spore-forming bacteria, and include the important human pathogens Clostridium 7 perfringens, Clostridium botulinum, and Clostridioides difficile, among numerous others. Clostridia have 8 been reported to form biofilms composed of cellulose, although the specific loci which encode the 9 cellulose synthase have not been identified. Here, we report the discovery of a gene cluster, which we 10 named ccsABZCD, among selected bacteria within class Clostridia that appears to encode a synthase 11 complex responsible for polymerization, modification, and export of an O-acetylated cellulose 12 exopolysaccharide. To test this hypothesis, we subcloned the putative glycoside hydrolase CcsZ and 13 solved the X-ray crystal structure of both apo-and product-bound CcsZ. Our results demonstrate that 14 CcsZ is in fact an endo-acting cellulase belonging to glycoside hydrolase family 5 . This is in 15 contrast to the Gram-negative cellulose synthase, which instead encodes BcsZ, a structurally distinct GH-16 8. We further show CcsZ is capable of hydrolysis of the soluble mock substrate carboxymethylcellulose 17 (CMC) with a pH optimum of 4.5. The data we present here serves as an entry point to an understanding 18 of biofilm formation among class Clostridia and allowed us to predict a model for Clostridial cellulose 19 synthesis.20 Author summary 21 Biofilms are communities of microorganisms that enmesh themselves in a protective matrix of elf-22 produced polysaccharide materials. Biofilms have demonstrated roles in both virulence and persistence 23 among bacterial pathogens of global health importance. The class Clostridia are a polyphyletic grouping 24 of primarily Gram-positive, anaerobic and spore-forming bacteria which contain the important and well-25 studied human pathogens Clostridioides difficile, Clostridium botulinum, and Clostridium perfringens, 26among others. Bacteria belonging to class Clostridia have been anecdotally reported to form biofilms described. In this work, we identify a gene cluster, which we name ccsABZHI, for the Clostridial cellulose 29 synthase, which bears remarkable similarity to molecular machinery required for the production of 30 cellulose biofilms in other Gram-negative bacteria. We further biochemically characterize one of these 31 enzymes, CcsZ, a predicted endoglucanase which we predicted from our model should cleave cellulose 32 exopolysaccharides. We show that CcsZ is in fact capable of this activity and belongs to a broader family 33 of glycoside hydrolases with unexpected...